This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To define the influence of stromal cells on trophoblast differentiation from human embryonic stem cells. PROGRESS: Studies of early placental development in humans are difficult because of limitations on experimental material availability from the perimplantation period. We utilized a three-dimensional co-culture system to determine the effects of various effector cell types on trophoblast differentiation. EGFP-expressing H1 human embryonic stem cells (H1EGFP hESC) were used in co-suspension with red Cell Tracker-labeled human term placental fibroblasts (TPF), chorionic villus sampling fibroblasts (CVS), or dermal fibroblasts (CI2F) to form combination embryoid bodies (EBs), with the goal of recapitulating placental morphogenesis through incorporation of placental mesenchymal cells. These EBs were maintained in suspension culture for 30 days. Media were collected daily for hormone assay, and EBs were fixed and processed for immunohistochemical staining to determine the number of cytokeratin and hCG- positive cells in the different treatment groups. Overall, the results demonstrated that when utilizing mesenchymal cells for EB preparation from term placentas (TPF), combination EB-derived trophoblasts secrete higher levels of hCG and progesterone compared to EBs made without effector cells. This is due to the secretory activity of the EB-derived trophoblasts and not due to the number of differentiated trophoblasts per EB, demonstrating that nontrophoblast cells of the placenta can influence trophoblast endocrine activity. This work used WNPRC Stem Cell Resources. PUBLICATIONS: Giakoumopoulos, M., Siegfried, L.M. Dambaeva, S.V., Garthwaite, M.A., and T.G. Golos. 2010 Placental-derived mesenchyme influences chorionic gonadotropin and progesterone secretion of human embryonic stem cell-derived trophoblasts. Reproductive Sciences. 17(9):798-808. PMID: 20601539.